Induction of microsomal stearyl coenzyme A desaturase in the newly hatched chicks.

The development of the stearyl-CoA desaturase system was studied in newly hatched chicks. The desaturation activity was very low in hepatic microsomes from chick embryos, less than 0.05 nmol of oleate formed min^?1 (mg of protein)^?1. After hatching and feeding, the desaturation activity gradually increased to 4–5 nmol of oleate formed min^?1 (mg of protein)^?1 in 6-day-old chicks. This increase could be prevented by administration of cycloheximide or actinomycin D. Measurement of the microsomal electron transfer components throughout the induction period showed no significant changes in the NADH- or NADPH-specific reductases or in the concentrations of cytochromes b₅ and P-450. However, the activity of the terminal component of the desaturase system (the desaturase enzyme) increased in parallel with the desaturation activity. Supplementing the liver microsomes from chick embryos with isolated desaturase enzyme resulted in the formation of an active desaturation system. It is proposed that the induction of the stearyl-CoA desaturase system during development of newly hatched chicks is dependent on the synthesis of the terminal desaturase enzyme.     

Properties of a Δ5-fatty acyl-CoA desaturase in the cotyledons of developing Limnanthes alba

A Δ5-fatty acid desaturase activity in extracts of developing meadowfoam (Limnanthes alba) seeds was characterized. Since the system was unstable after fractional centrifugation, only the cell-free homogenate was employed in these studies. Maximal enzyme activity was observed at pH 8. A time-course analysis of the substrates and products of the reaction suggested that the Δ5 desaturation occurs on an acyl-CoA. The desaturase activity was stimulated by n-octyl-glucopyranoside, catalase, ferredoxin, and several other compounds. Inhibition was observed with Triton X-100, p-chloromercuribenzoate, dithiothreitol, cysteine, and several other compounds. The enzyme was not inhibited by carbon monoxide (95%) suggesting that it is not associated with cytochrome P-450. Eicosanoyl-CoA saturated the enzyme at 1.5 μm and was inhibitory at higher concentrations. The desaturase had an apparent K_m of 23 μm for O₂. NADH was a better electron donor than NADPH. Among the potential substrates tested, the enzyme preferred eicosanoyl-CoA, but also desaturated palmitoyl-CoA, stearoyl-CoA, and eicosanoyl-ACP at fairly high rates.     

Chronic ethanol administration decreases fatty acyl-CoA desaturase activities in rat liver microsomes

The Δ⁹-desaturase system in liver microsome from rats treated chronically with ethanol was studied. Stearoyl-CoA desaturase activity decreased by 80% and palmitoyl-CoA desaturase activity was not detectable in microsomes from ethanol-fed rats, while activities of electron transport components such as NADH-cytochrome c and NADH-ferricyanide reductases remained unchanged. However, chronic ethanol administration resulted in an adaptive induction of the activity of NADPH-cytochrome c reductase and the contents of cytochrome b₅ and P-450. The activity of the terminal component (cyanide-sensitive factor; CSF) of the desaturase system was greatly depressed by ethanol treatment. The NADH/NAD ratio in microsomes of ethanol-fed rats increased over 2-fold. These results suggest that, during chronic ethanol ingestion, decreased activities of Δ⁹-desaturases are due mainly to a decreased content of the terminal component of the desaturase system.     

Stimulation of hepatic cytochrome b5-mediated lipid desaturation by renal microsomes

Although microsomes prepared from rat kidney cortex contained significant concentrations of both NADH cytochrome b₅ reductase and cytochrome b₅, they did not catalyze cytochrome b₅-dependent Δ⁹ oxidative lipid desaturation. However, incubation of kidney microsomes in the presence of control liver microsomes resulted in a two-fold increase in fatty acid desaturase activity over that seen with liver microsomes alone. Addition of kidney microsomes to liver microsomes prepared from animals maintained on a fat free diet resulted in an increased desaturase activity which was twice that seen with the control liver preparation. Kidney microsomes alone did not catalyze the cytochrome P-450-dependent N-demethylation of aminopyrine, and in contrast to the desaturate, no increase in demethylase activity was observed when kidney microsomes were added to liver microsomes.     

The effect of hypolipidemic drugs WY14643 and DH990, and lysophospholipids on the metabolism of oleate in plants

The effects of the addition of hypolipidemic drugs and 1-acylglycerolipids on the metabolism of oleate in plants have been studied in vivo and in vitro. Using aged potato slices with [¹⁴C]oleate as a precursor, it was found that these drugs markedly inhibited both the incorporation into complex lipids and the desaturation of oleate to linoleate. Moreover, in vitro experiments, carried out with microsomes prepared from developing safflower seeds and [¹⁴C]oleate or [¹⁴C]oleoyl-CoA as precursors, confirmed the inhibitory effect of the drugs on oleate desaturation, and showed that while WY14643 mainly affected oleoyl thiokinase activity, DH990 exerted its strongest effect on the formation of PL, indicating that the mode of action of these two drugs in safflower microsomes is essentially different. Addition of LPC or LPE stimulated the incorporation of radiolabeled precursor into PC and PE, respectively, as well as the desaturation of oleate to linoleate when [¹⁴C]oleoyl-CoA was the precursor. The evidence obtained suggests that oleoyl-PE, as well as oleoyl-PC, should be considered as a possible substrate for oleate desaturation in plants.     

Fatty acid desaturase system of yeast microsomes. Involvement of cytochrome b5-containing electron-transport chain.

The oxidative desaturation of palmitoyl CoA by microsomes from anaerobically grown Saccharomyces cerevisiae has been studied by using NADH as electron donor. The desaturation product was identified as palmitoleic acid by periodate oxidation. The desaturase activity was sensitive to relatively high concentrations of cyanide; the concentration of cyanide causing half-maximal inhibition was determined to be 7.1 mm. The rate of reoxidation of cytochrome b₅ in NADH-reduced microsomes was stimulated by the addition of palmitoyl CoA, and the amount of cytochrome b₅ reoxidized by the palmitoyl CoA added could be closely correlated to the amount of palmitoleate formed. No stimulation of the reoxidation of cytochrome b₅ was induced by palmitoyl CoA in microsomes prepared from the desaturase-repressed cells and from a desaturase-deficient mutant, strain KD-20. It is concluded that the fatty acyl CoA desaturase system of yeast microsomes involves cytochrome b₅ as an electron carrier and that the terminal desaturase is sensitive to relatively high concentrations of cyanide.     

Immunochemical evidence for the participation of cytochrome b5 in microsomal stearyl-CoA desaturation reaction

A rabbit antiserum was prepared against rat liver microsomal cytochrome b₅, and utilized in demonstrating the participation of this cytochrome in the microsomal stearyl-CoA desaturation reaction. The antiserum inhibited the NADH-cytochrome c reductase activity of rat liver microsorncs, but it did not inhibit either NADH-ferricyanide or NADPH-cytochrome c reductase activity of the microsomes. Thus, the inhibitory effect of the antiserum on the microsomal electron-transferring reactions seemed to be specific to those which require the participation of cytochrome b₅.The NADH-dependent and NADPH-dependent desaturations of stearyl CoA by rat liver microsomes were strongly inhibited by the antiserum. The reduction of cytochrome b₅ by NADH-cytochrome b₅ reductase as well as the reoxidation of the reduced cytochrome b₃ by the desaturase, the terminal cyanide-sensitive factor of the desaturation system, was also strongly inhibited by the antiserum. When about 90%, of cytochrome b₅ was removed from rat liver microsomes by protease treatment, the desaturation activity of the microsomes became much more sensitive to inhibition by the antiserum. These results confirmed our previous conclusion that the reducing equivalent for the desaturation reaction is transferred from NAD(P)H to the cyanidesensitive factor mainly via cytochrome b₅ in the microsomal membranes.     

Cooperative interaction of reduced pyridine nucleotides with estrogen synthetase of human placental microsomes

The estrogen synthetase present in human placental microsomes appears to be dependent on the cooperative interaction of the reduced cofactors NADPH and NADH for optimal activity. Using steady-state concentrations of either cofactor, it was found that while the estrogen synthetase activity followed hyperbolic saturation kinetics with NADPH (K_mapp = 14 μM), the enzyme followed sigmoidal saturation kinetics when the cofactor was NADH, with the half-maximum velocity attained at a cofactor concentration of 1.1 mm. The maximum velocity obtained with NADPH as the cofactor was greater than with corresponding concentrations of NADH. Estrogen synthetase activity in the presence of NADH was not due to NADPH contamination. NADH, in the presence of small concentrations of NADPH (0.5 to 5 μm), stimulated significantly the rate of estrogen formation from androstenedione by placental microsomes and, in addition, the enzyme saturation kinetics changed from sigmoidal to hyperbolic, thus mimicking the effect of NADPH. Estrogen synthetase activity, measured in the presence of 1 mm NADH, was stimulated in a dose-dependent manner by NADPH (K_mapp = 0.4 μM NADPH) and, when the enzyme was measured in the presence of 5 μm NADPH, the activity was stimulated in a dose-dependent manner by NADH (K_mapp = 45 μM NADH). Estrogen synthetase activity measured in the presence of NADH, without and with NADPH (1 μm) remained linear both with time of incubation for approximately 15 min and with microsomal protein concentration up to 3 mg/ml. The apparent K_m of estrogen synthetase for androstenedione, when measured in the presence of NADH, was 1 μm. The synergistic interaction between NADH and NADPH in stimulating placental estrogen synthetase activity observed in vitro may, conceivably, take place in vivo in the intact placenta.     

Identification of a soluble protein stimulator of plasmalogen biosynthesis and stearoyl-coenzyme a desaturase

Stimulation of the desaturation of 1-alkyl-2-acyl-sn-glycero-3-phosphoethanolamine (GPE), which forms ethanolamine plasmalogens, by a component of the 105,000g supernatant has been previously reported. We have isolated the stimulatory protein and identified it as catalase. Purified rat liver catalase or commercial bovine liver catalase is as effective in stimulating microsomal 1-alkyl-2-acyl-GPE desaturation as the soluble proteins. The stimulatory effect of these proteins is eliminated by catalase inhibitors. It appears that catalase stimulates the desaturation of 1-alkyl-2-acyl-GPE by preventing inactivation of the enzyme system by H₂O₂ or a decomposition product of H₂O₂. The cytochrome b₅ content and NADH oxidation are depressed in Fischer R-3259 sarcoma microsomes by H₂O₂; this effect is eliminated by catalase. However, since measurable inhibition of 1-alkyl-2-acyl-GPE desaturase by H₂O₂ still occurred in the presence of catalase, the inhibition by H₂O₂ cannot be explained solely on the basis of cytochrome b₅ inactivation. The desaturation of stearoyl-coenzyme A, a reaction analogous in many respects to 1-alkyl-2-acyl-GPE desaturation, was also found to be stimulated by catalase.     

Desaturation of Oleic and Linoleic Acids by Leaves of Dark- and Light-grown Maize Seedlings

Oleate and linoleate desaturation in leaves of maize seedlings was largely independent of previous light treatment of the seedlings; there was no evidence of light-induced desaturase activities. These results are in sharp contrast to those observed with developing cucumber cotyledons in which pronounced increase in desaturation occurs after exposure of tissue to light. The rates of desaturation of oleate were about four times those of linoleate in both etiolated and 16-hour greened maize leaves. In both etiolated and greened tissues, about two-thirds of the label from oleate was esterified after 4 hours, half of which was in phosphatidylcholine. Phosphatidylcholine and diglyceride contained large proportions of [¹⁴C]linoleate formed from [¹⁴C]oleate but not [¹⁴C]linolenate. In monogalactolipid, about two-thirds of the labeled fatty acids were linolenate. In vivo desaturase activity was present in tissue of widely different levels of differentiation and chlorophyll content obtained from light-grown maize seedlings.     

Author index

The stearoyl-coenzyme A desaturase system of L-M cells, grown as monolayers, was examined in microsomal membranes that contained 8.2% phosphatidylisopropylethanolamine, an unnatural phospholipid analog. Desaturation of both [1-¹⁴C]stearic acid by whole cells and [1-¹⁴C]stearoyl-coenzyme A by cell-free homogenates, or microsomes, was decreased to about 40% of control levels in cells that had been grown for 24 h in the presence of 10 mmN-isopropylethanolamine. No decrease in microsomal NADH- or NADPH-dependent cytochrome c reductase activities or the level of cytochrome b₅ was found in the L-M cells that had been treated for 24 h with N-isopropylethanolamine. Although amino acid transport into L-M cells was not affected by treatment with N-isopropylethanolamine, protein synthesis was decreased by about 30%. These results indicate that the decrease in stearoyl-coenzyme A desaturation in the modified membranes is specifically associated with the terminal oxidase activity (cyanide-sensitive factor) of the desaturase enzyme complex.     

Acyl CoA:Cholesterol acyl transferase activity in human placental microsomes: Inhibition by progesterone

The characteristics of acyl CoA:cholesterol acyltransferase (ACAT; EC 2.3.1.26) in microsomes prepared from human term placenta were studied and the rate of incorporation of [1-¹⁴C] oleoyl CoA into cholesteryl esters was measured. The apparent K_m of the enzyme for [1-¹⁴C] oleoyl CoA was 38 ± 9 μm and the V for the reaction was 15 ± 6 pmol × mg^? protein × min^?1. The Hill coefficient for the reaction was 1.2, indicative of some degree of positive cooperativity. Cholesterol, added to the incubation mixture, did not influence ACAT activity, indicating that endogenous microsomal cholesterol served as an effective substrate for the placental ACAT enzyme. However, [1,2-³H]cholesterol in the presence of oleoyl CoA was incorporated into cholesteryl esters by placental microsomes. When progesterone was present in the incubation mixture at a concentration of 20 μm, ACAT activity was inhibited 50%. Pregnenolone, 5α-dihydroprogesterone, 17α-hydroxyprogesterone, deoxycorticosterone, dehydroisoandrosterone, androstenedione, testosterone, and estradiol-17β also inhibited ACAT activity, whereas corticosterone, cortisol, and estriol had little effect. These results are supportive of the view that ACAT activity in human placenta may be regulated by endogenously synthesized steroid hormones.     

Some properties of a microsomal oleate desaturase from leaves.

1. When [1-14C]oleoyl-CoA was incubated with a pea-leaf homogenate oleate was both incorporated into microsomal 3-sn-phosphatidylcholine and released as the unesterified fatty acid. The proportion of oleate incorporated into this phospholipid was dependent on the relative amounts of thiol ester and microsomal preparation present in reactions. 2. At the concentrations of microsomal preparation and [14C]oleoyl-CoA used to study oleate desaturation the metabolism of the thiol ester was essentially complete after 5 min incubation, but the loss of label from 3-sn-phosphatidylcholine oleate and the concomitant increase in radioactivity in the linoleate of this phospholipid proceeded at approximately linear rates over a 60 min period. The kinetics of labelling of unesterified linoleate was consistent with the view that this labelled fatty acid was derived from 3-sn-phosphatidylcholine. 3. Oleate desaturation required oxygen and with unwashed microsomal fractions was stimulated either by NADPH or by the 105 000g supernatant. Washed microsomal preparations did not catalyse desaturation, but actively was restored by the addition of NADPH, 105 000G supernatant or Sephadex-treated supernatant. NADPH could be replaced by NADH or NADP+, but not by NAD+. 4. Microsomal fractions from mature and immature maize lamina and expanding spinach leaves also rapidly incorporated oleate from ([14C]oleoyl-CoA into 3-sn-phosphatidylcholine, but desaturation of 3-sn-phosphatidylcholine oleate was detected only with microsomal preparations from immature maize lamina. 5. It is proposed that leaf microsomal preparations posses an oleate desaturase for which 3-sn-phosphatidylcholine oleate is either the substrate or an immediate precursor of the substrate.     

Characterization of fatty acid desaturase activity in rat lung microsomes.

Preparations of rat lung microsomes containing 0.030-0.050 nmole of cytochromes P-450 and b5 per mg microsomal protein have been observed to contain significant levels of fatty acid desaturase activity. Both stearoyl CoA and palmitoyl CoA are desaturated to their monounsaturated analogues, oleic acid and palmitoleic acid, respectively. Activity (per mg microsomal protein) of the lung preparations varied according to the diet of the animals prior to killing in the order: fat free diet greater than normal rat chow greater than starvation. All preparations exhibited approximately 50% inhibition when incubated in the presence of 0.10 mM CN-. Maximal activity was obtained with the 0.50 mM NADH less activity with equal amounts of NADPH, and there was no synergistic interaction of NADH and NADPH together. The rate of desaturation was linear with protein concentrations between 0.15-1.5 mg microsomal protein/incubation at incubation times up to 8 min. A pH optimum range of 7.0-7.4 was observed. For all variables of fatty acid desaturase activity which were examined, the rate of desaturation of stearoyl CoA was approximately twice that for palmitoyl CoA. These results indicate that the same fatty acid desaturation system which is functional in the liver is also present in significant amounts in mammalian lungs.     

Oleate metabolism in microsomes from developing leaves ofPisum sativum L.

Microsomes from young leaves of pea,Pisum sativum L., metabolized oleate principally by the reactions mediated by oleoyl-CoA synthetase, oleoyl-CoA thioesterase, oleoyl-CoA: phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase. Hydrogen peroxide specifically inhibited oleate desaturation and the evidence presented argues for a specific inhibition of the terminal enzyme of the desaturase system, i.e. oleoyl phosphatidylcholine desaturase. Catalase, ascorbic acid, or ascorbate peroxidase, in conjunction with ascorbic acid, stimulated oleate desaturation, possibly by the removal of hydrogen peroxide. Lysophosphatidylcholine was found to be the preferred acceptor for acyl transfer from oleoyl-CoA, which indicates that the transfer of oleoyl moieties was catalyzed predominantly by oleoyl-CoA:lysophosphatidylcholine acyltransferase. Acyl exchange between oleoyl-CoA and phosphatidylcholine, with a possible involvement of phospholipases, was also detected but at much lower rates than acyl transfer. When intact or broken chloroplasts were added to microsomes, which had been preincubated with oleoyl-CoA, some stimulation of the reactions catalyzed by oleoyl-CoA:phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase was observed. However, only minor amounts of microsomal linoleoyl phosphatidylcholine were converted to galactolipids containing linolenoyl moieties.Abbreviations FA unesterified fatty acid (s) - PC phosphatidylcholines - 18:1 oleoyl moieties - 18:2 lmoleoyl moieties Dedicated to Professor Helmut K. Mangold, Bundesanstalt für Fettforschung, Münster, on his 60th birthday     

Assay for the terminal enzyme of the stearoyl coenzyme A desaturase system using chick embryo liver microsomes.

The NADH-dependent stearoyl CoA desaturase of hepatic microsomes (EC 1.14.99.5) is an enzyme system consisting of cytochrome b5 reductase (EC 1.6.2.2), cytochrome b5, and the terminal desaturase. We have developed a simple method for routine assay of the terminal enzyme based on complementation of the enzyme with chick embryo liver microsomes lacking desaturase activity. Desaturation of [1-14C]stearoyl CoA by the enzyme-microsome mixture is then assayed by thin-layer chromatography of the reaction products and determination of the amount of oleate formed. Microsomes from the livers of starved-refed rats were used as the source of the stearoyl CoA desaturase. The enzyme alone, solubilized and free from cytocrome b5 reductase and cytochrome b5, was unable to catalyze the desaturation of stearoyl CoA. However, after preincubation with chick embryo liver microsomes in the presence of 1% Triton X-100, the enzyme was active. The enzyme activity was linear with time and desaturase protein under the conditions described and depended on the concentrations of Triton X-100 present in the preincubation and the assay. The optimum concentrations of Triton X-100 were 1% for the preincubation and 0.1-0.15% in the assay. The desaturation activity was dependent on NADH and O2, and was inhibited 95% by 1 mM KCN. The use of chick embryo liver microsomes in this method eliminates the need to use purified cytochrome b5 reductase, cytochrome b5, and liposomes for routine assays and greatly reduces the complexities of timing and order of addition encountered in the existing assays.     

Δ5 Desaturase activity in rat kidney microsomes

Rat kidney microsomal fraction is able to catalyze the enzymatic desaturation of eicosatrienoic acid (20:3n-6) to arachidonic acid (20:4n-6) by the 5 desaturase pathway, in the presence of reduced nicotinamide adenine dinucleotide (NADH), adenosinetriphosphate (ATP) and coenzyme A (CoA). The substrate of the reaction [1-¹⁴C]eicosa-8,11,14trienoic acid (20:3n-6), was separated from the product [1-¹⁴C]eicosa-5,8,11,14-tetraenoic acid (20:4n-6) by reverse phase high-pressure liquid chromatography (RP-HPLC). These fatty acids were individually collected by monitoring the eluent at 205 nm and their radioactivity was measured by liquid scintillation counting. The 5 desaturase activity in kidney microsomes increased linearly with the substrate concentration up to 20 M. Enzymatic activity was sensitive to pH with the maximum at 7.0 and was proportional with incubation time up to 10 min. The apparent Km and Vmax of 5 desaturase were 56 M and 60 pmoles·min^–1·mg^–1 microsomal protein, respectively. Neither the cytosolic renal fraction nor the cytosolic liver fraction enhanced the 5 desaturase activity. Contrary to a report but in accordance to others, the present results suggest that rat kidneys can synthesize arachidonic acid at least to satisfy partially their needs for eicosanoid production.     

Degradation of Hepatic Stearyl CoA Δ9-Desaturase

Δ⁹-Desaturase is a key enzyme in the synthesis of desaturated fatty acyl-CoAs. Desaturase is an integral membrane protein induced in the endoplasmic reticulum by dietary manipulations and then rapidly degraded. The proteolytic machinery that specifically degrades desaturase and other short-lived proteins in the endoplasmic reticulum has not been identified. As the first step in identifying cellular factors involved in the degradation of desaturase, liver subcellular fractions of rats that had undergone induction of this enzyme were examined. In livers from induced animals, desaturase was present in the microsomal, nuclear (P-1), and subcellular fractions (P-2). Incubation of desaturase containing fractions at physiological pH and temperature led to the complete disappearance of the enzyme. Washing microsomes with a buffer containing high salt decreased desaturase degradation activity. N-terminal sequence analysis of desaturase freshly isolated from the P-1 fraction without incubation indicated the absence of three residues from the N terminus, but the mobility of this desaturase preparation on SDS-PAGE was identical to the microsomal desaturase, which contains a masked N terminus under similar purification procedures. Addition of concentrated cytosol or the high-salt wash fraction did not enhance the desaturase degradation in the washed microsomes. Extensive degradation of desaturase in the high-salt washed microsomes could be restored by supplementation of the membranes with the lipid and protein components essential for the reconstituted desaturase catalytic activity. Lysosomotrophic agents leupeptin and pepstatin A were ineffective in inhibiting desaturase degradation. The calpain inhibitor, N-acetyl-leucyl-leucyl-methional, or the proteosome inhibitor, Streptomyces metabolite, lactacystin, did not inhibit the degradation of desaturase in the microsomal or the P-1 and P-2 fractions. These results show that the selective degradation of desaturase is likely to be independent of the lysosomal and the proteosome systems. The reconstitution of complete degradation of desaturase in the high-salt–washed microsomes by the components essential for its catalytic activity reflects that the degradation of this enzyme may depend on a specific orientation of desaturase and intramembranous interactions between desaturase and the responsible protease.     

Inhibitory effects of alkyl gallate and its derivatives on fatty acid desaturation

Alkyl gallate, which is known as an antioxidant, intensively inhibited Δ5 and Δ6 desaturation in both rat liver microsomes and an arachidonic acid-producing fungus Mortierella alpina 1S-4. The rat liver microsomal Δ5 and Δ6 desaturases were inhibited by gallic acid esterified with alcohols with various numbers of carbons, suggesting that the necessary structure in an esterified alcohol for the inhibition is not so strict. Among the three hydroxy groups in gallic acid, the m-hydroxy group was shown to be the necessary structure. Kinetic analyses revealed that propyl gallate is a noncompetitive inhibitor of Δ5 desaturase (K_i = 2.6 · 10⁻⁵ M) and Δ6 desaturase (K_i = 1.7 · 10⁻⁴ M). These data indicate that alkyl gallate is a new type of desaturase inhibitor and different from known natural inhibitors, i.e., sesamin and curcumin.     

Functional expression of Brassica juncea oleate desaturase gene (Bjfad2) in Escherichia coli

The synthesis of polyunsaturated fatty acids, the most abundant fatty acids in plants, begins with a reaction catalyzed by fatty acid desaturase-2 (FAD2; EC 1.3.1.35), also called as microsomal Δ12 oleate desaturase. The gene (Bjfad2; GenBank accession No. EF639848) coding for this enzyme from Brassica juncea was previously isolated and characterized. However, functional identity of Bjfad2 was not established. Utilizing the known Bjfad2 cDNA sequence, the ORF of Bjfad2 gene was cloned into the pMAL C2X Escherichia coli expression vector and produced recombinant plasmid by insertion of isolated ORF downstream to the maltose-binding protein coding sequence. The pMALC2X-Bjfad2 vector was used to transform the TB1 strain of E. coli. Induced expression of pMAL-BJFAD2 fused product resulted in the synthesis of a polypeptide with an apparent molecular mass of 80 kDa, which was 8 kDa less than calculated mass as determined by SDS-PAGE, since the fused MalE-Bjfad2 gene contains eight additional codons located between the MalE and Bjfad2 gene. In vitro activity assay of oleate desaturase using the corresponding bacterial crude extracts confirmed that the polypeptide was the product of the Bjfad2 gene. The reaction products analysis of the fatty acid methyl esters by gas chromatography showed the presence of a new peak with a similar retention time to linoleic acid, which was absent in the control activity assay without electron donors. Thus, B. juncea gene has been functionally identified since it encodes the enzyme that catalyzed the desaturation of oleate to linoleate.